Hydrophobically modified antisense oligonucleotides comprising a ketal group

ABSTRACT

The present invention concerns an oligonucleotide modified by substitution at the 3′ or the 5′ end by a moiety comprising at least one ketal functional group, wherein the ketal carbon of said ketal functional group bears two saturated or unsaturated, linear or branched, hydrocarbon chains comprising from 1 to 22 carbon atoms, and the use therefore as a medicament, in particular for use for treating cancer.

The present invention concerns hydrophobically modified oligonucleotides, their process of manufacture, pharmaceutical compositions comprising said modified oligonucleotides, and their use as vehicles for the administration of drugs.

There is currently substantial interest in the use of nucleic acids for modifying gene expression for therapeutic purposes. The use of oligonucleotide (ON) analogues as potential therapeutic agents for modulating the expression of specific genes has shown promise for different classes of molecules, including aptamers, triplex-forming oligonucleotides, antisense oligonucleotides (ASO), small interfering RNAs (siRNAs) and antagomirs. However, one major barrier in the broad utilization of ON analogues as clinical drugs is their reduced ability to transverse cell membranes.

Cellular uptake and localization of ONs are crucial problems for their effects on the genetic expressions. Most approaches for their cellular delivery involve cationic lipophilic carriers and/or polymers. While such synthetic carriers can be exceptionally effective for delivering plasmid DNA into the cytoplasm of cells, most are of limited utility because of their toxicity to cells and poor efficiency in the case of ONs. Alternatively, a lipophilic moiety has been covalently tethered to the ON structures with the aim of improving cellular uptake and antisense activity. Lipid conjugated ONs (LONs) feature an oligonucleotide sequence as the polar head and at least one lipid moiety inserted either at the 3′- or 5′-end of the oligonucleotide or within the sequence. Interestingly, LONs self assemble to give aggregates such as micelles and vesicles. The appended lipidic segment of LONs brings about new properties to these surfactants like enhanced antisense activities, tagging of vesicles or lipid bilayers, and biological membranes. LONs have also been successfully implemented in original detection schemes for nucleic acids, and as biotechnological tools.

However, the LONs developed so far have the drawback of bearing non-cleavable lipid moieties which might affect the inhibitory efficiency of the ONs, once internalized in the targeted cells.

There is therefore still a need for alternative modified antisense ONs with (i) a high cellular uptake, (ii) a prolonged half-life on plasma, and (iii) a high efficiency of gene silencing in vivo.

The present invention arises from the unexpected finding by the inventors that an antisense oligonucleotide modified by substitution at the 5′ or 3′ end by a ketal moiety comprising two saturated linear hydrocarbon chains comprising 15 carbon atoms, which can be metabolized, had an increased inhibitory power in vivo compared to the same ASO bearing other 5′ modifications, even in the absence of any transfection reagent.

Additionally, the present inventors demonstrated that these LASOs were capable of aggregating thereby creating a lipophilic core or reservoir, which could be used for the loading of hydrophobic drugs, such as Paclitaxel, and that this drug loading could be modulated via the hybridization of the complementary ASO sequence. Such LASOs aggregates can therefore be used to specifically trigger the release of a drug in vivo.

The present invention therefore concerns an oligonucleotide modified by substitution at the 3′ or the 5′ end by a moiety comprising at least one ketal functional group, wherein the ketal carbon of said ketal functional group bears two saturated hydrocarbon chains comprising from 1 to 22 carbon atoms.

The present invention also relates to a process of manufacture of a modified oligonucleotide as defined herein, comprising the steps of:

-   -   (i) synthesizing the oligonucleotide;     -   (ii) modifying the oligonucleotide by reaction with a suitable         reactant comprising a ketal functional group comprising two         saturated or unsaturated, linear or branched, hydrocarbon chains         comprising from 1 to 22 carbon atoms;     -   (iii) recovering the modified oligonucleotide.

The present invention further concerns a modified antisense oligonucleotide as defined herein for use as a medicament, in particular for use for treating cancer.

The present invention also relates to an aqueous composition comprising modified oligonucleotides as defined herein, wherein the modified oligonucleotides self-assembled into micelles.

The present invention also concerns the use of this aqueous composition as a vehicle.

The present invention also relates to the aqueous composition defined herein, comprising an hydrophobic active principle hosted in said micelles, for use as a medicament, in particular for use for treating cancer.

DETAILED DESCRIPTION OF THE INVENTION Oligonucleotide

As used herein, the term “oligonucleotide” refers to a nucleic acid sequence, 3′-5′ or 5′-3′ oriented, which may be single- or double-stranded. The oligonucleotide used in the context of the invention may in particular be DNA or RNA.

The oligonucleotides used in the context of the invention may be further modified, preferably chemically modified, in order to increase the stability and/or therapeutic efficiency of the oligonucleotides in vivo. In particular, the oligonucleotide used in the context of the invention may comprise modified nucleotides.

Chemical modifications may occur at three different sites: (i) at phosphate groups, (ii) on the sugar moiety, and/or (iii) on the entire backbone structure of the oligonucleotide.

For example, the oligonucleotides may be employed as phosphorothioate derivatives (replacement of a non-bridging phosphoryl oxygen atom with a sulfur atom) which have increased resistance to nuclease digestion. 2′-methoxyethyl (MOE) modification (such as the modified backbone commercialized by ISIS Pharmaceuticals) is also effective.

Additionally or alternatively, the oligonucleotides of the invention may comprise completely, partially or in combination, modified nucleotides which are derivatives with substitutions at the 2′ position of the sugar, in particular with the following chemical modifications: O-methyl group (2′-O-Me) substitution, 2-methoxyethyl group (2′-O-MOE) substitution, fluoro group (2′-fluoro) substitution, chloro group (2′-Cl) substitution, bromo group (2′-Br) substitution, cyanide group (2′-CN) substitution, trifluoromethyl group (2′-CF₃) substitution, OCF₃ group (2′-OCF₃) substitution, OCN group (2′-OCN) substitution, O-alkyl group (2′-O-alkyl) substitution, S-alkyl group (2′-S-alkyl) substitution, N-alkyl group (2′-N-akyl) substitution, O-alkenyl group (2′-O-alkenyl) substitution, S-alkenyl group (2′-S-alkenyl) substitution, N-alkenyl group (2′-N-alkenyl) substitution, SOCH₃ group (2′-SOCH₃) substitution, SO₂CH₃ group (2′-SO₂CH₃) substitution, ONO₂ group (2′-ONO₂) substitution, NO₂ group (2′-NO₂) substitution, N₃ group (2′-N₃) substitution and/or NH₂ group (2′-NH₂) substitution.

Additionally or alternatively, the oligonucleotides of the invention may comprise completely or partially modified nucleotides wherein the ribose moiety is used to produce locked nucleic acid (LNA), in which a covalent bridge is formed between the 2′ oxygen and the 4′ carbon of the ribose, fixing it in the 3′-endo configuration. These constructs are extremely stable in biological medium, able to activate RNase H and form tight hybrids with complementary RNA and DNA.

Accordingly, in a preferred embodiment, the oligonucleotide used in the context of the invention comprises modified nucleotides selected from the group consisting of LNA, 2′-OMe analogs, 2′-phosphorothioate analogs, 2′-fluoro analogs, 2′-Cl analogs, 2′-Br analogs, 2′-CN analogs, 2′-CF₃ analogs, 2′-OCF₃ analogs, 2′-OCN analogs, 2′-O-alkyl analogs, 2′-S-alkyl analogs, 2′-N-alkyl analogs, 2′-O-alkenyl analogs, 2′-S-alkenyl analogs, 2′-N-alkenyl analogs, 2′-SOCH₃ analogs, 2′-SO₂CH₃ analogs, 2′-ONO₂ analogs, 2′-NO₂ analogs, 2′-N₃ analogs, 2′-NH₂ analogs and combinations thereof. More preferably, the modified nucleotides are selected from the group consisting of LNA, 2′-OMe analogs, 2′-phosphorothioate analogs and 2′-fluoro analogs.

Additionally or alternatively, some nucleobases of the oligonucleotide may be present as desoxyriboses. That modification should only affect the skeleton of the nucleobase, in which the hydroxyle group is absent, but not the side chain of the nucleobase which remains unchanged. Such a modification usually favors recognition of the iRNA by the DICER complex.

The oligonucleotide according to the invention may for example correspond to antisense oligonucleotides or to interfering RNAs (including siRNAs, shRNAs, miRNAs, dsRNAs, and other RNA species that can be cleaved in vivo to form siRNAs), that preferably target mRNAs of interest.

As used herein, an oligonucleotide that “targets” an mRNA refers to an oligonucleotide that is capable of specifically binding to said mRNA. That is to say, the oligonucleotide comprises a sequence that is at least partially complementary, preferably perfectly complementary, to a region of the sequence of said mRNA, said complementarity being sufficient to yield specific binding under intra-cellular conditions.

As immediately apparent to the skilled in the art, by a sequence that is “perfectly complementary to” a second sequence is meant the reverse complement counterpart of the second sequence, either under the form of a DNA molecule or under the form of a RNA molecule. A sequence is “partially complementary to” a second sequence if there are one or more mismatches.

Preferably, the oligonucleotide of the invention targets an mRNA encoding Translationally-Controlled Tumor Protein (TCTP), and is capable of reducing the amount of TCTP in cells.

Nucleic acids that target an mRNA encoding TCTP may be designed by using the sequence of said mRNA as a basis, e.g. using bioinformatic tools. For example, the sequence of SEQ ID NO: 5 can be used as a basis for designing nucleic acids that target an mRNA encoding TCTP.

Preferably, the oligonucleotides according to the invention are capable of reducing the amount of TCTP in cells, e.g. in cancerous cells such as LNCaP or PC3 cells. Methods for determining whether an oligonucleotide is capable of reducing the amount of TCTP in cells are known to the skilled in the art. This may for example be done by analyzing TCTP protein expression by Western blot, and by comparing TCTP protein expression in the presence and in the absence of the oligonucleotide to be tested (see FIG. 8 and Example 4).

The oligonucleotides according to the invention typically have a length of from 12 to 50 nucleotides, e.g. 12 to 35 nucleotides, from 12 to 30, from 12 to 25, from 12 to 22, from 15 to 35, from 15 to 30, from 15 to 25, from 15 to 22, from 18 to 22, or about 19, 20 or 21 nucleotides.

The oligonucleotides according to the invention may for example comprise or consist of 12 to 50 consecutive nucleotides, e.g. 12 to 35, from 12 to 30, from 12 to 25, from 12 to 22, from 15 to 35, from 15 to 30, from 15 to 25, from 15 to 22, from 18 to 22, or about 19, 20 or 21 consecutive nucleotides of a sequence complementary to the mRNA of SEQ ID NO: 5.

In particular, the inventors have identified thirteen oligonucleotides targeting an mRNA encoding TCTP that are very efficient in reducing the amount of TCTP in cells. These oligonucleotides target the regions consisting of nucleotides 153 to 173 of SEQ ID NO: 5, nucleotides 220 to 240 of SEQ ID NO: 5, nucleotides 300 to 320 of SEQ ID NO: 5, and nucleotides 320 to 340 of SEQ ID NO: 5, respectively. All of these oligonucleotides target the translated region of the TCTP mRNA (which extends from nucleotide 94 to 612 of SEQ ID NO: 5).

Therefore, the oligonucleotides according to the invention preferably target a sequence overlapping with nucleotides 153 to 173, or with nucleotides 221 to 240 or with nucleotides 300 to 340 of SEQ ID NO: 5, said oligonucleotide being a DNA or a RNA. Such an oligonucleotide may for example target:

-   -   a sequence consisting of nucleotides 153 to 173 or of         nucleotides 221 to 240 or of nucleotides 300 to 320, or of         nucleotides 320 to 340 of SEQ ID NO: 5, or     -   a sequence comprised within nucleotides 153 to 173 or within         nucleotides 221 to 240, or within nucleotides 300 to 320, or         within nucleotides 320 to 340 of SEQ ID NO: 5, or     -   a sequence partially comprised within nucleotides 153 to 173 or         within nucleotides 221 to 240, or within nucleotides 300 to 320,         or within nucleotides 320 to 340 of SEQ ID NO: 5.

The oligonucleotides according to the invention may for example comprise a fragment of at least 10 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO: 2 (5′-ACCAATGAGCGAGTCATCAA-3′), SEQ ID NO: 3 (5′-AACCCGUCCGCGAUCUCCCGG-3′), SEQ ID NO: 6 (5′-AACTTGTTTCCTGCAGGTGA-3′), SEQ ID NO: 7 (5′-TGGTTCATGACAATATCGAC-3′), SEQ ID NO: 8 (5′-TAATCATGATGGCGACTGAA-3′), SEQ ID NO: 16 (5′-ACCAGTGATTACTGTGCTTT-3′), SEQ ID NO: 17 (5′-CTTGTAGGCTTCTTTTGTGA-3′), SEQ ID NO: 18 (5′-ATGTAATCTTTGATGTACTT-3′), SEQ ID NO: 19 (5′-GTTTCCCTTTGATTGATTTC-3′), SEQ ID NO: 20 (5′-TTCTGGTCTCTGTTCTTCAA-3′), SEQ ID NO: 25 (5′-AGAAAATCATATATGGGGTC-3′), SEQ ID NO: 27 (5′-TTAACATTTCTCCATTTCTA-3′), SEQ ID NO: 29 (5′-GTCATAAAAGGTTTTACTCT-3′) and SEQ ID NO: 31 (5′-GAAATTAGCAAGGATGTGCT-3′). More preferably, the oligonucleotides comprise a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29 and SEQ ID NO: 31. The oligonucleotides according to the invention may for example comprise a fragment of at least 10 consecutive nucleotides of a sequence of SEQ ID NO: 2 or of a sequence of SEQ ID NO: 6, or of a sequence of SEQ ID NO: 7, or of a sequence of SEQ ID NO: 3. Most preferably, they comprise a sequence of SEQ ID NO: 2, or a sequence of SEQ ID NO: 6, or a sequence of SEQ ID NO: 7 or a sequence of SEQ ID NO: 3. Still preferably, the oligonucleotides according to the invention comprise a fragment of at least 10 consecutive nucleotides of a sequence of SEQ ID NO: 6. Most preferably, they comprise a sequence of SEQ ID NO: 6.

In a preferred embodiment according to the invention, the oligonucleotide is an antisense oligonucleotide.

As used herein, the term “antisense oligonucleotide” refers to a single stranded DNA or RNA with complementary sequence to its target mRNA, and which binds its target mRNA thereby preventing protein translation either by steric hindrance of the ribosomal machinery or induction of mRNA degradation by ribonuclease H.

The antisense oligonucleotide may be a DNA or a RNA molecule.

Said antisense oligonucleotide may for example comprise or consist of a fragment of at least 10, 12, 15, 18 or 20 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO: 2 (5′-ACCAATGAGCGAGTCATCAA-3′), SEQ ID NO: 6 (5′-AACTTGTTTCCTGCAGGTGA-3′), SEQ ID NO: 7 (5′-TGGTTCATGACAATATCGAC-3′), SEQ ID NO: 8 (5′-TAATCATGATGGCGACTGAA-3′), SEQ ID NO: 16 (5′-ACCAGTGATTACTGTGCTTT-3′), SEQ ID NO: 17 (5′-CTTGTAGGCTTCTTTTGTGA-3′), SEQ ID NO: 18 (5′-ATGTAATCTTTGATGTACTT-3′), SEQ ID NO: 19 (5′-GTTTCCCTTTGATTGATTTC-3′), SEQ ID NO: 20 (5′-TTCTGGTCTCTGTTCTTCAA-3′), SEQ ID NO: 25 (5′-AGAAAATCATATATGGGGTC-3′), SEQ ID NO: 27 (5′-TTAACATTTCTCCATTTCTA-3′), SEQ ID NO: 29 (5′-GTCATAAAAGGTTTTACTCT-3′) and SEQ ID NO: 31 (5′-GAAATTAGCAAGGATGTGCT-3′), preferably of a sequence SEQ ID NO: 2 (5′-ACCAATGAGCGAGTCATCAA-3′), or of a sequence of SEQ ID NO: 6 (5′-AACTTGTTTCCTGCAGGTGA-3′), or of a sequence of SEQ ID NO: 7 (5′-TGGTTCATGACAATATCGAC-3′). Preferably, it comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29 and SEQ ID NO: 31, more preferably, from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 7. Most preferably, it comprises or consists of the sequence SEQ ID NO: 6.

In another preferred embodiment according to the invention, the oligonucleotide is an interfering RNA (iRNA).

RNA interference is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNA (dsRNA) can block gene expression when it is introduced into worms (Fire et al., 1998, Nature 391:806-811). dsRNA directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. RNA interference involves mRNA degradation, but many of the biochemical mechanisms underlying this interference are unknown. The use of RNA interference has been further described in Carthew et al. (2001, Current Opinions in Cell Biology, 13:244-248) and in Elbashir et al. (2001, Nature, 411:494-498). The iRNA molecules of the invention are double-stranded or single-stranded RNA, preferably of from about 21 to about 23 nucleotides, which mediate RNA inhibition. That is, the iRNA of the present invention preferably mediate degradation of mRNA encoding TCTP.

The term “iRNA” include double-stranded RNA, single-stranded RNA, isolated RNA (partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA), as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the RNA or internally (at one or more nucleotides of the RNA). Nucleotides in the iRNA molecules of the present invention can also comprise non-standard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides. Collectively, all such altered iRNA compounds are referred to as analogs or analogs of naturally-occurring RNA. iRNA of the present invention need only be sufficiently similar to natural RNA that it has the ability to mediate RNA interference. As used herein the phrase “mediate RNA Interference” refers to and indicates the ability to distinguish which mRNA are to be affected by the RNA interference machinery or process. RNA that mediates RNA interference interacts with the RNA interference machinery such that it directs the machinery to degrade particular mRNAs or to otherwise reduce the expression of the target protein. In one embodiment, the present invention relates to iRNA molecules that direct cleavage of specific mRNA to which their sequence corresponds. It is not necessary that there be perfect correspondence of the sequences, but the correspondence must be sufficient to enable the iRNA to direct RNA interference inhibition by cleavage or lack of expression of the target mRNA.

The iRNA molecules of the present invention may comprise an RNA portion and some additional portion, for example a deoxyribonucleotide portion. The total number of nucleotides in the RNA molecule is suitably less than 49 in order to be effective mediators of RNA interference. In preferred RNA molecules, the number of nucleotides is 16 to 29, more preferably 18 to 23, and most preferably 21-23.

As indicated above, the term “iRNA” includes but is not limited to siRNAs, shRNAs, miRNAs, dsRNAs, and other RNA species that can be cleaved in vivo to form siRNAs.

A “short interfering RNA” or “siRNA” comprises a RNA duplex (double-stranded region) and can further comprise one or two single-stranded overhangs, 3′ or 5′ overhangs.

A “short hairpin RNA (shRNA)” refers to a segment of RNA that is complementary to a portion of a target gene (complementary to one or more transcripts of a target gene), and has a stem-loop (hairpin) structure.

“MicroRNAs” or “miRNAs” are endogenously encoded RNAs that are about 22-nucleotide-long, that post-transcriptionally regulate target genes and are generally expressed in a highly tissue-specific or developmental-stage-specific fashion. One can design and express artificial miRNAs based on the features of existing miRNA genes. The miR-30 (microRNA 30) architecture can be used to express miRNAs (or siRNAs) from RNA polymerase II promoter-based expression plasmids (Zeng et al, 2005, Methods enzymol. 392:371-380). In some instances the precursor miRNA molecules may include more than one stem-loop structure. The multiple stem-loop structures may be linked to one another through a linker, such as, for example, a nucleic acid linker, a miRNA flanking sequence, other molecules, or some combination thereof.

In a preferred embodiment according to the invention, the iRNA comprises or consists of a fragment of at least 10, 12, 15 or 18 consecutive nucleotides of a sequence of SEQ ID NO: 3 (5′-AACCCGUCCGCGAUCUCCCGG-3′). Preferably, such a nucleic acid is an iRNA comprising or consisting of a sequence of SEQ ID NO: 3. Most preferably, such a nucleic acid is a siRNA or a shRNA. The sequence of SEQ ID NO: 3 may be modified, and e.g. correspond to a modified sequence of SEQ ID NO: 3 such as a 5′-AACCCGUCCGCGAUCUCCCdGdG-3′ sequence.

The present inventors also demonstrated that modified oligonucleotides comprising an oligonucleotide consisting of an adenine 15-mer of sequence SEQ ID NO: 32 (dA₁₅) or of a thymidine 15-mer of sequence SEQ ID NO: 33 (dT₁₅) were particularly useful to form micelles capable of encapsulating drugs.

Accordingly, in a preferred embodiment, the oligonucleotide is selected from the group consisting of dT₁₅, dA₁₅, and an oligonucleotide consisting of the sequence SEQ ID NO: 6.

Lipid Conjugate

The present invention concerns an oligonucleotide, as defined in the section “Oligonucleotide” herein above, modified by substitution at the 3′ or the 5′ end by a moiety comprising at least one ketal functional group, wherein the ketal carbon of said ketal functional group bears two saturated or unsaturated, preferably saturated, linear or branched, preferably linear, hydrocarbon chains comprising from 1 to 22 carbon atoms, preferably from 6 to 20 carbon atoms, in particular 10 to 19 carbon atoms, and even more preferably from 12 to 18 carbon atoms.

In a preferred embodiment according to the invention, the modified oligonucleotide is of the general formula (I):

wherein:

-   -   Oligo represents an oligonucleotide sequence which may be         oriented 3′-5′ or 5′-3′, simple and/or double stranded, ADN,         ARN, and/or comprise modified nucleotides, in particular an         oligonucleotide as defined in the section “Oligonucleotide”         herein above;     -   X represents a divalent linker moiety selected from ether —O—,         thio —S—, amino —NH—, and methylene —CH₂—;     -   R₁ and R₂ may be identical or different and represent:         -   (i) a hydrogen atom,         -   (ii) a halogen atom, in particular fluorine atom,         -   (iii) a hydroxyl group,         -   (iv) an alkyl group comprising from 1 to 12 carbon atoms;     -   L₁ and L₂ may be identical or different and represent a         saturated or unsaturated, preferably saturated, linear or         branched, preferably linear, hydrocarbon chain comprising from 1         to 22 carbon atoms, preferably from 6 to 20 carbon atoms, more         preferably from 12 to 18 carbon atoms,     -   B is an optionally substituted nucleobase, selected from the         group consisting of purine nucleobases, pyrimidine nucleobases,         and non-natural monocyclic or bicyclic heterocyclic nucleobases         wherein each cycle comprises from 4 to 7 atoms.

In the context of the invention, the term “alkyl” refers to a hydrocarbon chain that may be a linear or branched chain, containing the indicated number of carbon atoms. For examples, C₁-C₁₂ alkyl indicates that the group may have from 1 to 12 (inclusive) carbon atoms in it.

Preferably, the oligonucleotide sequence “Oligo-” is connected to the divalent linker moiety X via a phosphate moiety —O—P(═O)(O)—, at its 3′ or 5′ end, advantageously at its 5′ end.

In a preferred embodiment according to the invention, the modified oligonucleotide is of the general formula (I′):

wherein:

-   -   wherein X, R₁, R₂, L₁, L₂ and B are as defined above in formula         (I),     -   [3′ - - - 5′] represents, along with the PO₃ ⁻ residue, an         oligonucleotide as defined in the section “Oligonucleotide”         herein above, and     -   A⁺ represents a cation, preferably H⁺, Na⁺, K⁺ or NH₄ ⁺.

In the formulae (I) and (I′), the divalent linker moiety is preferably ether —O—.

In the formulae (I) and (I′), R₁ and R₂ are preferably hydrogen atoms.

In a preferred embodiment according to the invention, the modified oligonucleotide is of the formula (I″):

wherein A⁺, X, L₁, L₂ and B are as defined above in formula (I) and [3′ - - - 5′] represents, along with the PO₃ ⁻ residue, an oligonucleotide as defined in the section “Oligonucleotide” herein above.

In the formulae (I), (I′) and (I″), L₁ and L₂ preferably represent a hydrocarbon chain, preferably a linear hydrocarbon chain, comprising from 6 to 22 carbon atoms, preferably from 8 to 18 carbon atoms, advantageously from 12 to 16 carbon atoms, more advantageously 15 carbon atoms.

In the formulae (I), (I′) and (I″), B preferably represents a non substituted nucleobase selected from the group consisting of uracil, thymine, adenine, guanine, cytosine, 6-methoxypurine, 7-methylguanine, xanthine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine and hypoxanthine. Preferably, in the formulae (I), (I′) and (I″), B represents a non substituted nucleobase selected from the group consisting of uracil, thymine, adenine, cytosine, 6-methoxypurine and hypoxanthine. More preferably, in the formulae (I), (I′) and (I″), B represents uracil.

In a preferred embodiment according to the invention, the modified oligonucleotide is of the formula (I′″):

wherein A⁺ is as defined above in formula (I) and [3′ - - - 5′] represents, along with the PO₃ ⁻ residue, an oligonucleotide as defined in the section “Oligonucleotide” herein above.

Process of Manufacture

The present invention also provides a process of manufacture of a modified oligonucleotide as defined hereabove, comprising the steps of:

-   -   (i) synthesizing the oligonucleotide, as defined in the section         “Oligonucleotide” herein above;     -   (ii) modifying the oligonucleotide by reaction with a suitable         reactant comprising a ketal functional group comprising two         saturated or unsaturated, preferably saturated, linear or         branched, preferably linear, hydrocarbon chains comprising from         1 to 22 carbon atoms, preferably from 6 to 20 carbon atoms, more         preferably from 12 to 18 carbon atoms;     -   (iii) recovering the modified oligonucleotide.

Step (ii) is generally carried out using a coupling methodology.

According to a preferred embodiment of the process of the invention, step (ii) is carried out using the phosphoramidite methodology, which is well-known for the synthesis of oligonucleotides.

Preferably, the reactant used in step (ii) comprises a phosphoramidite group.

A “phosphoramidite group” refers to a monoamide of a phosphite diester moiety, which can be represented as follows >N—P(—O—)₂.

The reactant used in step (ii) is preferably of formula (II):

wherein R₁, R₂, L₁, L₂ and B are as defined above in formula (I).

In the formula (II), R₁ and R₂ are preferably hydrogen atoms.

In the formula (II), L₁ and L₂ preferably represent a hydrocarbon chain, preferably a linear hydrocarbon chain, comprising from 6 to 22 carbon atoms, preferably from 8 to 18 carbon atoms, advantageously from 12 to 16 carbon atoms, more advantageously 15 carbon atoms.

In the formula (II), B preferably represents a non substituted nucleobase selected from the group consisting of uracil, thymine, adenine, guanine, cytosine, 6-methoxypurine, 7-methylguanine, xanthine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine and hypoxanthine. Preferably, in the formulae (I), (I′) and (I″), B represents a non substituted nucleobase selected from the group consisting of uracil, thymine, adenine, cytosine, 6-methoxypurine and hypoxanthine. More preferably, in the formulae (I), (I′) and (I″), B represents uracil.

When the reactant comprises a phosphoramidite group, step (ii) is generally carried out in the presence of a coupling agent, such as N-benzylthiotetrazole, which activates the phosphoramidite. The oligonucleotide synthesized in step (i), advantageously comprising an OH 5′ end, is then added, and the coupling occurs.

The modified oligonucleotide of the invention is then obtained according to usual procedures well-known in the framework of oligonucleotides synthesis.

Aqueous Compositions

The present invention also provides an aqueous composition comprising modified oligonucleotides as defined hereabove, wherein the modified oligonucleotides self-assembled into micelles.

A micelle is an aggregate of surfactant molecules dispersed in a liquid colloid. A typical micelle in aqueous solution forms an aggregate with the hydrophilic “head” regions of the molecules in contact with surrounding aqueous solvent, sequestering the hydrophobic “tail” regions of the molecules in the micelle centre.

The modified oligonucleotides self-assemble into micelles having a core/shell structure, wherein the shell is hydrophilic and is formed of the oligonucleotide parts of the modified oligonucleotides, and wherein the core is lipophilic and is formed of the saturated or unsaturated, linear or branched, hydrocarbon chains in C₁-C₂₂ of the modified oligonucleotides.

The aqueous compositions may comprise up to 50% by weight of modified oligonucleotides, preferably from 0.1% to 40%, in particular from 1% to 20%, and especially from 8% to 15% by weight of modified oligonucleotides.

According to a preferred embodiment of the aqueous composition of the invention, it further comprises a hydrophobic active principle hosted in said micelles.

Said micelles can be used for the loading of hydrophobic drugs. Loading of such micelles with hydrophobic drug can vary between 2 μM to 2 mM.

The hydrophobic active principle is preferably selected from the group consisting of Paclitaxel, Docetaxel, Vincristine, Vinorelbine, and Abraxane; Tamoxifen, Gonadotrophin-releasing hormone (GnRH) agonists and antagonists, androgen receptor (AR) antagonist, and estrogen receptor (ER) antagonists; Cyclophosphamide, Chlorambucil and Melphalan; Methotrexate, Cytarabine, Fludarabine, 6-Mercaptopurine and 5-Fluorouracil; Doxorubicin, Irinotecan, Platinum derivatives, Cisplatin, Carboplatin, Oxaliplatin; Bicalutamide, Anastrozole, Examestane and Letrozole; Imatinib (Gleevec), Gefitinib and Erlotinib; Rituximab, Trastuzumab (Herceptin) and Gemtuzumab ozogamicin; Interferon-alpha; Tretinoin and Arsenic trioxide; Bevicizumab, Serafinib and Sunitinib.

More preferably, the hydrophobic active principle is Paclitaxel.

Use as Vehicle

The present invention also concerns the use of an aqueous composition as defined in the section “Aqueous composition” hereabove as a vehicle.

As used herein, the term “vehicle” refers to a carrier of a medicinally and/or pharmaceutically active substance.

Preferably, the aqueous composition as defined in the section “Aqueous composition” hereabove is used as a carrier of sparingly hydrosoluble active principle.

Such a vehicle is notably useful for the administration of such active substance by way of intravenous, intraperitoneal, subcutaneous or oral routes, or direct hemoral injection.

The active principle is preferably selected from the group consisting of Paclitaxel, Docetaxel, Vincristine, Vinorelbine, and Abraxane; Tamoxifen, Gonadotrophin-releasing hormone (GnRH) agonists and antagonists, androgen receptor (AR) antagonist, and estrogen receptor (ER) antagonists; Cyclophosphamide, Chlorambucil and Melphalan; Methotrexate, Cytarabine, Fludarabine, 6-Mercaptopurine and 5-Fluorouracil; Doxorubicin, Irinotecan, Platinum derivatives, Cisplatin, Carboplatin, Oxaliplatin; Bicalutamide, Anastrozole, Examestane and Letrozole; Imatinib (Gleevec), Gefitinib and Erlotinib; Rituximab, Trastuzumab (Herceptin) and Gemtuzumab ozogamicin; Interferon-alpha; Tretinoin and Arsenic trioxide; Bevicizumab, Serafinib and Sunitinib.

More preferably, the hydrophobic active principle is Paclitaxel.

Medical Indications

The inventors have found that the siRNAs and antisense oligonucleotides targeting TCTP inhibit the castration-resistant progression of PC3, LNCaP and C4-2 cells and xenografts and enhance docetaxel and castration-sensitivity. Therefore, the present invention provides a modified oligonucleotide according to the invention as defined hereabove, in particular a modified antisense oligonucleotide according to the invention, in particular an oligonucleotide which targets an mRNA encoding TCTP, for use as a medicament, more particularly for use in the treatment or prevention of cancer, preferably hormone- and/or chemo-resistant cancer. The present invention also provides a modified oligonucleotide according to the invention as defined hereabove, in particular a modified antisense oligonucleotide according to the invention, more particularly an oligonucleotide which targets an mRNA encoding TCTP, for use in restoring sensibility to hormone- and/or chemo-therapy in a patient suffering from cancer.

The invention also provides the use of a modified oligonucleotide according to the invention as defined hereabove, in particular a modified antisense oligonucleotide according to the invention, in particular an oligonucleotide which targets an mRNA encoding TCTP, for the manufacture of a medicament, in particular for the manufacture of a medicament intended to treat or prevent cancer, preferably hormone- and/or chemo-resistant cancer. The invention also provides the use of a modified oligonucleotide according to the invention as defined hereabove, in particular a modified antisense oligonucleotide according to the invention, more particularly an oligonucleotide which targets an mRNA encoding TCTP, for the manufacture of a medicament intended to restore sensibility to hormone- and/or chemo-therapy in a patient suffering from cancer.

The invention also provides a method for treating or preventing cancer and/or for restoring sensibility to hormone- and chemo-therapy comprising the step of administering an effective amount of a modified oligonucleotide according to the invention as defined hereabove, in particular a modified antisense oligonucleotide according to the invention, more particularly an oligonucleotide which targets an mRNA encoding TCTP, to an individual in need thereof.

As used herein, the term “cancer” refers to any type of malignant (i.e. non benign) tumor. The tumor may correspond to a solid malignant tumor, which includes e.g. carcinomas, adenocarcinomas, sarcomas, malignant melanomas, mesotheliomas, blastomas, or to a blood cancer such as leukaemias, lymphomas and myelomas. The cancer may for example correspond to an advanced prostate cancer, a pancreatic cancer, a bladder cancer, an ovarian cancer, a testicular cancer, a cortical adenoma, a colon cancer, a colorectal cancer, a breast cancer or a liver cancer.

Cancers which are preferably treated according to the invention are those wherein TCTP is expressed at higher levels in cancerous cells than in non-cancerous cells of the same tissue type. Exemplary such cancers include without limitation prostate cancer, colon cancer, colorectal cancer, breast cancer, liver cancer, erythroleukemia, gliomas, melanomas, hepatoblastomas and lymphomas.

The oligonucleotides according to the invention, in particular the oligonucleotides which target an mRNA encoding TCTP, are believed to be capable of delaying or preventing the emergence of a resistant hormone-independent phenotype, and to be capable of reversing a resistant hormone-independent phenotype. They are thus particularly suitable for use in the treatment of a hormone-independent cancer or of a hormone-dependent cancer in which hormone-independency is expected to occur. The term “castration” in the expression “castration-resistant” or “castration-independency” according to the invention refers to “hormone” and corresponds to “hormone-resistant” or “hormone-independency”. Androgen independency refers to a hormone-independency. Indeed, an androgen-independent prostate cancer (AIPC) is a castration-resistant prostate cancer (CRPC).

Therefore, a preferred embodiment is directed to an oligonucleotide according to the invention as defined hereabove, in particular an oligonucleotide which targets an mRNA encoding TCTP, for use in the treatment or prevention of a hormone-independent or chemo-resistant cancer.

Since the oligonucleotides according to the invention, in particular the oligonucleotides which target an mRNA encoding TCTP, are capable of restoring sensitivity to drugs, they are particularly suitable for use in the treatment of advanced cancers or chemotherapy resistant cancers. The oligonucleotides according to the invention, in particular the oligonucleotides which target an mRNA encoding TCTP, can notably be used as a second line therapy.

The skilled in the art is capable of determining whether a cancer is an “advanced” cancer using well-known classification methods, such as e.g. the grade or the TNM classification. For example, the grade (G1-4) of the cancer cells may be used. More specifically, cancer cells are “low grade” if they appear similar to normal cells, and “high grade” if they appear poorly differentiated. For example, G3 or G4 cancers would be classified as advanced cancers. Additionally or alternatively, the TNM classification may be used. In this classification, T(a,is,(0),1-4) indicates the size or direct extent of the primary tumor, N(0-3) indicates the degree of spread to regional lymph nodes, and M(0/1) indicates the presence of metastasis. For example, a T4/N3/M1 cancer would be classified as an advanced cancer.

Most preferably, the cancer is a prostate cancer, e.g. an advanced prostate cancer and/or an androgen-independent prostate cancer. In a specific embodiment, androgen-dependent prostate cancers can be excluded from the scope of the cancers to be treated in the frame of the present invention.

The inventors have further found that the antisense oligonucleotides according to the invention enhanced the anticancer effects of chemotherapy by docetaxel both in vitro and in vivo. In particular, the antagonists according to the invention are believed to be capable of reversing a castration-resistant phenotype and of restoring sensitivity to drugs. Therefore, the oligonucleotides according to the invention, in particular the oligonucleotides which target an mRNA encoding TCTP, can advantageously be used (simultaneously or sequentially) in combination with at least one second anti-cancer agent (e.g. in the frame of a chemotherapy).

In particular, the oligonucleotides according to the invention, in particular the oligonucleotides which target an mRNA encoding TCTP, may be used in the frame of a combination chemotherapy. The oligonucleotides according to the invention, in particular the oligonucleotides which target an mRNA encoding TCTP, may for example be used (simultaneously or sequentially) in combination with at least one of the following anti-cancer agents:

-   -   an antimitotic agent such as Docetaxel, Vincristine, Paclitaxel         (Taxol), Vinorelbine, and Abraxane;     -   a hormonal therapy drug, such drugs being commonly used in the         frame of treatment of hormone-sensitive cancers. Hormonal         therapy drugs include, e.g., Tamoxifen, Gonadotrophin-releasing         hormone (GnRH) agonists and antagonists, androgen receptor (AR)         antagonist, and estrogen receptor (ER) antagonists;     -   an alkylating agent such as Cyclophosphamide, Chlorambucil and         Melphalan;     -   an antimetabolite such as Methotrexate, Cytarabine, Fludarabine,         6-Mercaptopurine and 5-Fluorouracil;     -   a topoisomerase inhibitor such as Doxorubicin, Irinotecan,         Platinum derivatives, Cisplatin, Carboplatin, Oxaliplatin;     -   an aromatase inhibitor such as Bicalutamide, Anastrozole,         Examestane and Letrozole;     -   a signaling inhibitor such as Imatinib (Gleevec), Gefitinib and         Erlotinib;     -   a monoclonal antibody such as Rituximab, Trastuzumab (Herceptin)         and Gemtuzumab ozogamicin;     -   a biologic response modifier such as Interferon-alpha;     -   a differentiating agent such as Tretinoin and Arsenic trioxide;         and/or     -   an agent that block blood vessel formation (antiangiogenic         agents) such as Bevicizumab, Serafinib and Sunitinib.

In addition, the method of treating or preventing cancer according to the invention may be associated with a radiation therapy, surgery and/or androgen withdrawal.

Administration of oligonucleotides according to the invention, in particular of oligonucleotides which target an mRNA encoding TCTP, can be carried out using various mechanisms known in the art, including naked administration and administration in pharmaceutically acceptable lipid carriers or nanoparticles. For example, lipid carriers for antisense delivery are disclosed in U.S. Pat. Nos. 5,855,911 and 5,417,978.

They may be for example administered by intravenous, intraperitoneal, subcutaneous or oral routes, or direct local tumor injection.

The oligonucleotide is administered in an “effective amount”, i.e. in an amount sufficient to treat or prevent the cancer. It will be appreciated that this amount will vary both with the effectiveness of the oligonucleotides or other therapeutic agent employed, and with the nature of any carrier used. The determination of appropriate amounts for any given composition is within the skill in the art, through standard series of tests designed to assess appropriate therapeutic levels.

In the frame of the present invention, the individual preferably is a human individual. However, the veterinary use of the antagonist according to the present invention is also envisioned. The individual may thus also correspond to a non-human individual, preferably a non-human mammal.

The term “treating” is meant a therapeutic method, i.e. a method aiming at curing, improving the condition and/or extending the lifespan of an individual suffering from the cancer.

By “preventing” is meant a prophylactic method, e.g. aiming at reducing the risk of relapse and/or reducing the risk of appearance of a hormone-independency.

Prostate cancer is one cancer that overexpresses TCTP in advanced cancers, and in particular in cancers that have become castration-resistant. For treatment of prostate cancer, the oligonucleotides according to the invention, in particular the oligonucleotides which target an mRNA encoding TCTP, are suitably administered after initial of androgen withdrawal. Initiation of androgen withdrawal may be accomplished via surgical (removal of both testicles) or medical (drug-induced suppression of testosterone) castration, which is currently indicated for treatment of prostate cancer. Medical castration can be achieved by various regimens, including LHRH agents or antiandrogens (see e.g. Cleave et al., 1999, CMAJ 160:225-232). Intermittent therapy in which reversible androgen withdrawal can be performed as described in Cleave et al. (1998, Eur. Urol. 34: 37-41).

The inhibition of TCTP, in particular of TCTP expression by the oligonucleotides according to the invention, may be transient. For treatment of prostate cancer, the TCTP inhibition should ideally occur coincident with androgen withdrawal. In humans, this means that TCTP inhibition should be effective starting within a day or two of androgen withdrawal (before or after) and extending for about 3 to 6 months. This may require multiple doses to accomplish. It will be appreciated, however, that the period of time may be more prolonged, starting before castration and expending for substantial time afterwards without departing from the scope of the invention.

The present inventors also demonstrated that the modified oligonucleotides of the invention were capable of forming micelles encapsulating hydrophobic active principles, which could release their content when hybridizing with their complementary sequences (FIGS. 2 and 3). Accordingly, the aqueous compositions of the invention are useful to specifically deliver active principles to cells of interest in order to treat a disease.

The present invention thus also provides an aqueous composition as defined in the section “Aqueous composition” hereabove, for use as a medicament, in particular for use for treating cancer, as defined above.

The present invention also provides the use of an aqueous composition as defined in the section “Aqueous composition” hereabove, for the manufacture of a medicament, in particular intended to treat or prevent cancer, as defined above.

The present invention also provides a method for treating or preventing cancer, as defined above, comprising the step of administering an effective amount of an aqueous composition according to the invention as defined in the section “Aqueous composition” hereabove, to an individual in need thereof.

Pharmaceutical Compositions

The present invention also provides a pharmaceutical composition comprising a modified oligonucleotide according to the invention, and a pharmaceutically acceptable carrier.

Pharmaceutical compositions formulated in a manner suitable for administration of oligonucleotides are known to the skilled in the art. For example, lipidic carriers (in particular liposomes) are particularly suitable pharmaceutically acceptable carriers when administering an oligonucleotide according to the invention.

Non-limiting examples of pharmaceutically acceptable carriers suitable for formulation with the oligonucleotide of the instant invention include: PEG conjugated nucleic acids, phospholipid conjugated nucleic acids, nucleic acids containing lipophilic moieties, phosphorothioates, P-glycoprotein inhibitors (such as Pluronic P85) which can enhance entry of drugs into various tissues, biodegradable polymers (such as poly (DL-lactide-coglycolide) microspheres) for sustained release delivery after implantation, and loaded nanoparticles (such as those made of polybutylcyanoacrylate).

The invention also features pharmaceutical composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, branched and unbranched or combinations thereof, or long-circulating liposomes or stealth liposomes). Oligonucleotides of the invention can also comprise covalently attached PEG molecules of various molecular weights. These formulations offer a method for increasing the accumulation of drugs in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al., 1995, Chem. Rev., 95:2601-2627; Ishiwata et al., 1995, Chem. Pharm. Bull., 43:1005-1011). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., 1995, Science, 267:1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238:86-90). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA. Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes.

The pharmaceutical composition of the invention may comprise stabilizers, buffers, and the like. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions of the present invention may be formulated and used as tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions or suspensions for injectable administration.

The choice of the formulation ultimately depends on the intended way of administration, such as e.g. an intravenous, intraperitoneal, subcutaneous or oral way of administration, or a local administration via tumor injection. Preferably, the pharmaceutical composition according to the invention is a solution or suspension, e.g. an injectable solution or suspension. It may for example be packaged in dosage unit form.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 shows the sequence of human TCTP protein.

SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 to SEQ ID NO: 31 show ASO sequences.

SEQ ID NO: 3 shows the sequence of a TCTP siRNA according to the invention.

SEQ ID NO: 4 shows the sequence of a scramble oligonucleotide that has been used as a control.

SEQ ID NO: 5 shows the sequence of a human TCTP mRNA. The regions that are targeted by the TCTP ASO of SEQ ID NO: 2 and by the TCTP siRNA of SEQ ID NO: 3 are indicated.

SEQ ID NO: 32 shows the sequence of dA₁₅.

SEQ ID NO: 33 shows the sequence of dT₁₅.

SEQ ID NO: 34 shows the sequence of the non-complementary single-strand DNA used in Example 2.

The invention will now be described more in detail by way of the examples below and the drawings in annex.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the structural formula of a LON comprising a ketal functional group according to the present invention.

FIG. 2 shows a schematic representation of single and double strand LON forming micelles with different drug loading.

FIG. 3 shows a schematic representation of controlled drug release using single and double strand LON based micelles.

FIG. 4 shows images obtained by transmission electronic microscopy (TEM) (A), and scanning electronic microscopy (SEM) (B) of ²LON-A₁₅ (C=40 μM in water).

FIG. 5 shows the release of Paclitaxel from ²LON-T₁₅ micelles (200 μM based on the LON) (left bar), after treatment with non complementary cDNA (middle bar) and dA15 (right bar) (10 μM phosphate buffer pH 7, 100 mM NaCl). The mean size of the micellar aggregates measured (by Dynamic Light Scattering in the absence of Paclitaxel) is indicated on top for each of the oligonucleotides systems.

FIG. 6 shows LON-assisted aqueous solubilization of Paclitaxel in 1 mM phosphate buffer and 100 mM NaCl. Oligonucleotide duplexes (when considered) were annealed prior to the addition of Paclitaxel.

FIG. 7 shows TCTP and GAPDH protein expression in prostatic cells transfected, in the presence of oligofectamine with (A) TCTP ASO#15PD or TCTP LASO#15PD, or with scramble Scr#PD and LScr#PD serving as negative controls, (B) with TCTP ASO#15PTO or TCTP LASO#15PTO, or with scramble Scr#PTO and LScr#PTO serving as negative controls, 2 days after transfection, and (C) with LASO#15LNA or LScr#LNA serving as negative control, 3 days after transfection.

FIG. 8 shows TCTP and GAPDH protein expression, 3 days after transfection, in prostatic cells transfected, in the absence of oligofectamine with (A) TCTP ASO#15PTO or TCTP LASO#15PTO, or with scramble Scr#PTO and LScr#PTO serving as negative controls and (B) with LASO#15LNA or LScr#LNA serving as negative control.

FIG. 9 shows TCTP protein expression, 6 days after transfection, in prostatic cells transfected, in the presence or absence of Oligofectamine with TCTP ASO#15PTO or TCTP LASO#15PTO, or with scramble Scr#PTO and LScr#PTO serving as negative control.

FIG. 10 shows TCTP protein expression, 3 days after transfection, in prostatic cells transfected, in the presence or absence of Oligofectamine with TCTP FASO#15 or with scramble FASO#Scr serving as negative control.

EXAMPLES Example 1

This example describes the synthesis of the ketal phosphoramidite.

Preparation of the Nucleoside Amphiphile Phosphoramidite 1:

The steps required for the synthesis of the phosphoraramidite synthon are indicated schematically below.

2′,3′-O-16-hentriacontanyliden-uridine 1′.

Palmitone (1 g, 2.22 mmol) was added to a mixture of dry THF (20 mL), uridine (2.70 g, 11.11 mmol.), TsOH (0.42 g, 2.22 mmol) and triethylorthoformate (1.85 mL, 11.11 mmol.). The solution was refluxed for 1 day, quenched with triethylamine (0.6 mL) and poured into a solution of H₂O (50 mL), ice (50 g) and NaHCO₃ (4 g) then stirred for 15 min. The organic phase was dried over Na₂SO₄ and concentrated in vacuo. The remaining solid was triturated in MeOH to give compound 1′ (0.74 g, 49%) as a white powder.

¹H NMR (300.13 MHz, CDCl₃): 0.87 (t, J=6.6 Hz, 6H, 2CH₃), 1.24 (m, 48H, 24CH₂), 1.41 (m, 4H, 2CH₃CH₂), 1.56 (m, 2H, CCH₂), 1.73 (m, 2H, CCH₂), 3.05 (m, 1H, OH), 3.80 (dd, J=12.0 Hz, J=3.6 Hz, 1H, H_(5′)), 3.91 (dd, J=12.0 Hz, J=2.7 Hz 1H, H_(5′)), 4.28 (m, 1H, H_(4′)), 4.95 (dd, J=6.6 Hz, J=3.6 Hz, 1H, H_(3′)), 5.04 (dd, J=6.6 Hz, J=2.7 Hz, 1H, H_(2′)), 5.55 (d, J=2.7 Hz, 1H, H_(1′)), 5.72 (d, J=8.1 Hz, 1H, CH), 7.33 (d, J=8.1 Hz, 1H, CH), 9.43 (m, 1H, NH). ¹³C NMR (75.47 MHz, CDCl₃): 14.10 (CH₃), 22.70 (CH₂), 23.54 (CH₂), 24.15 (CH₂), 29.33-29.81 (CH₂), 31.89 (CH₂), 36.85 (CH₂C00), 36.98 (CH₂COO), 62.65 (C_(5′)), 80.29 (C_(4′)), 83.84 (C_(3′)), 87.29 (C_(2′)), 96.34 (C_(1′)), 102.56 (CH), 118.39 (OCO), 143.16 (CH), 150.32 (CO), 163.33 (CO). FAB MS ([M+H]⁺=677).

Phosphoramidite 1.

Compound 1′ was dried over P₂O₅ overnight under reduced pressure before use. Diisopropylethylamine (0.052 mL, 0.299 mmol), 1′ (97 mg, 0.149 mmol) and 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite were dissolved in 3 mL dichloromethane and the solution stirred at room temperature for 1 h. Sodium bicarbonate 0.1M (3 mL) was poured into the flask and the aqueous phase extracted with dichloromethane. Flash chromatography (Hex/AcOEt/TEA 70/25/5) afforded 90 mg (69%) of the desired phosphoramidite as a white solid.

¹H NMR (300.13 MHz, CDCl₃): 0.88 (t, J=6.6 Hz, 6H, 2CH₃), 1.19 (m, 12H, iPr), 1.27 (m, 50H, 25 CH₂), 1.43 (m, 2H, CH₂), 1.56 (m, 2H, CH₂), 1.74 (m, 2H, CH₂), 2.65 (m, 2H, iPr), 3.60 (m, 2H, CH₂CN), 3.85 (m, 4H, OCH_(2′), H_(5′)), 4.36 (m, 1H, H_(4′)), 4.83 (m, 2H, H_(2′,3′)), 5.72 (d, J=8.1 Hz, 1H, CH), 5.81 (s, 1H, H_(1′), dia1), 5.90 (s, 0.5H, H_(1′), dia2), 7.48 (d, J=8.1 Hz, 0.5H, CH, dia1), 7.57 (d, J=8.1 Hz, 0.5H, CH, dia2). ESI MS ([M−H]⁻=876).

Example 2

In this example, LONs were investigated for their ability to self assemble and load Paclitaxel. In order to optimize the inclusion of the lipophilic Paclitaxel, LONs were synthesized with either 2 hydrocarbon chains attached at their respective 5′ end (FIG. 1). The inventors showed that LON based micelles, which are efficient host for hydrophobic drugs like Paclitaxel, can provide stimuli-responsive drug-carriers sensitive to the presence of a complementary oligonucleotide.

Materials and Methods

1. LON Synthesis and MS

LONs were synthesized using the phosphoramidite methodology on an automated Expedite 8909 DNA synthesizer at the μmole scale on 500 Å primer support (loading: 60-100 μmol/g, Link technologies, Synbase Control Pore Glass). The elongation of the oligonucleotide can be achieved following both a 3′ to 5′ and 5′ to 3′ elongation procedures. Accordingly, the nucleolipid modified phosphoramidite is inserted at the last position (either 5′ or 3′, depending on the elongation procedure selected). Autoclaved milliQ water was used whenever water was required for the handling of oligonucleotides or LONs.

Mass spectra measurements were performed on a MALDI-Tof-ToF mass spectrometer (Ultraflex, Bruker Daltonics, Bremen, Germany). Best results were obtained in the linear mode with positive-ion detection. Mass spectra were acquired with an ion source voltage 1 of 25 kV, an ion source voltage 2 of 23.5 kV, a lens voltage of 6 kV, by accumulating the ion signals from 1000 laser shots at constant laser fluence with a 100 Hz laser. External mass calibration was achieved using a mixture of oligonucleotides dT₁₂-dT₁₈ (Sigma).

1:1 mixture of samples of LONs (˜20-50 μM) and matrix was spotted on a MALDI target and air-dried before analysis.

The performance of the following matrices was evaluated: 2,5-dihydroxybenzoic acid (DHB), 2,4,6-trihydroxy-acetophenone (THAP), 3-hydroxypicolinic acid (3-HPA) and 2,6-dihydroxyacetophenone (DHA). THAP at a concentration of 20 mg/mL in a 4:1 mixture of ethanol and 100 mM aqueous ammonium citrate was shown to yield optimal mass spectral results.

2. Transmission and Scanning Electron Microscopy (TEM and SEM)

Transmission Electron Microscopy (TEM).

Samples were imaged by negative staining microscopy with a Hitachi H 7650 electron microscope. Samples containing LONs were transferred to a carbon-coated copper grid for ten minutes. The sample was then dried and stained with 2.5% (W/W) of uranyl acetate in water for five minutes.

Scanning Electron Microscopy (SEM).

Samples were examined at an accelerating voltage of 10 kV. Typically 20 ml of sample containing the LON aggregates were dried spread on polished stainless steel platelets and then coated with a 6-10 nm layer of gold-palladium alloy under vacuum with a sputter Coater Balzers SCD050. Observations were made with a Philips XL30S FEG instrument.

3. Solubilization of Paclitaxel in Aqueous Solutions of LONs

Representative example: Paclitaxel solubilization by a mixture of dA₁₅ and 2LON T₁₅ oligonucleotides.

Preparation of the Samples.

For the experiments of LON-assisted solubilization of Paclitaxel, 40 nmol of dA₁₅ and ²LON-T₁₅ were dried (speedvac). 0.2 mL PBS (10 mM phosphate buffer pH 7+100 mM of NaCl) were then added and the mixture vortexed.

The obtained aqueous solution was added to a test tube containing 0.6 mg of thoroughly dried Paclitaxel. Vortexing and then sonication at 30° C. for 30 min produced turbid colloidal suspensions (pure buffer solutions gave clear solutions above the Paclitaxel precipitate). Subsequent centrifugation (2000 rpm; 20 min) followed by filtration through a 450 nm filter gave the sample ready for HPLC analysis.

For the release of Paclitaxel experiments, the procedure was similar to the one described above except that dA₁₅ was not added in the first place. After the ¹LON T₁₅+Paclitaxel solution had been filtered, solution was added to an equimolar amount of dry dA15 or control oligonucleotide. The mixture was vortexed and left standing at room temperature for 8 h before the suspension was filtered again. The long period of time left for the hybridization of the 2 oligonucleotides was necessary to leave time for the released Paclitaxel molecules to aggregate in large filterable particles.

Determination of Paclitaxel Concentrations.

The concentrations of Paclitaxel in the different aqueous solutions were measured by reverse phase HPLC (Macherey Nagel EC 250/4 Nucleosil 120-5 C4) with detection at 227 nm. Elution conditions (eluent A (V/V): 5% CH₃CN/95% 0.1 M TEAA pH 7; eluent B: 80% CH₃CN/20% 0.1 M TEAA pH 7): isochratic (16% of B) for 2 min, 16→90% of B after 28 min and back to 16% of B.

The Paclitaxel concentrations were estimated by measuring the area of the Paclitaxel peak in the chromatogram after injection of a fixed volume (45 μL) of the solution. The latter was translated into a Paclitaxel concentration using a calibration curve. The concentrations tested for the calibration curve spanned the range of Paclitaxel concentrations obtained in the Paclitaxel-loading experiments.

Results and Discussion

1. Chemistry of LONs.

LONS were efficiently synthesized using the classical phosphoramidite chemistry for oligonucleotides. ²LON-A₁₅ and ²LON-T₁₅ were synthesized via the phosphoramidite 1 derived from the palmitone ketal of uridine 1′ of example 1

2. Physicochemical Properties of LONs.

As true surfactants, LONs can self assemble in aqueous media. Both the lipophilic segment and the oligonucleotide sequence were found to markedly influence the aggregation behavior of the LONs of the invention. Overall and not surprisingly, adenosine-containing LONs (²LON-A₁₅) were less soluble in water than their thymidine counterparts (²LON-T₁₅). If ²LON-T₁₅ aqueous solutions did not show any sign of precipitation after HPLC purification and storage, ²LON-A₁₅ was only sparingly soluble in water (up to ca. 50 μM). Yet, addition of a buffer (PBS or AcOH/TEA) resulted in a better solubilisation of ²LON-A₁₅. Dynamic light scattering, transmission electron microscopy and SEM experiments were carried out with the solutions of these 2 purified LONs. Aqueous samples containing LONs were studied under different conditions including pure water and aqueous suspensions of varying ionic strength and temperature (25-45° C.). All the LONs investigated namely ²LON-T₁₅ and ²LON-A₁₅ yielded small spherical objects ranging from approximately 8 to 20 nm in diameter as characterized by DLS, transmission and scanning electronic microscopies (TEM and SEM). For example, TEM and SEM imaging of ²LON-A₁₅ samples (C=40 μM) dissolved in pure water showed micellar aggregates (FIGS. 4A and 4B). The micellar architecture of these assemblies can be explained based upon the molecular dimensions of 15 nucleobase-long LONs. Indeed, such molecules are flexible, with cone shape structure and dimensions compatible with the self-aggregation into spherical micelles. The DLS study of ²LON-T₁₅ in water indicates that the size of aggregates is poorly sensitive to ionic strength. Likewise, the size of aggregates is poorly affected by temperature in the 25-45° C. range or the concentration of the amphiphile.

3. Drug Loading Experiments.

Physicochemical experiments and microscopy observations demonstrated that LONs self-associate into micelle-like aggregates. The inventors hypothesized that the hydrophobic core of LON aggregates could promote the solubilization in aqueous environments of hydrophobic drugs like Paclitaxel. To this end, the LONs were solubilized (200 or 300 μM solutions) in 10 mM PBS buffer at pH 7 and 100 mM NaCl. The LON aqueous solutions were bath-sonicated and heated in the presence of a thin film of Paclitaxel and the resulting suspensions filtered through a 0.4 μm pore to remove non-incorporated Paclitaxel aggregates. The Paclitaxel concentrations still remaining in solution were then determined by RP-C₄-HPLC (FIG. 6). The oligonucleotide sequence was quite unexpectedly found to be of prime importance (compare results II.1 and II.4 in FIG. 6): ²LON-A₁₅ turned out to be almost twice as potent as ²LON-T₁₅ at solubilizing Paclitaxel. The unmodified dA₁₅ and dT₁₅ oligonucleotides being both very poor at solubilizing Paclitaxel, cooperativity somehow exists between the dA₁₅ and the alkyl segments of ²LON-A₁₅ to host more drug molecules. Besides, when ²LON-A₁₅ and ²LON-T₁₅ complementary single strand DNA (cDNA: dT₁₅ and dA₁₅ respectively) were allowed to hybridize before the Paclitaxel solubilizing step, the Paclitaxel intake clearly decreases with both ²LON-A₁₅.dT₁₅ and ²LON-T₁₅.dA₁₅ aggregates compared to single-strand (ss) LON (compare results II.1/II.2 and II.4/II.5 respectively). If this decrease remained modest (37%) for ²LON-T₁₅.dA₁₅ aggregates, it reached 61% for ²LON-A₁₅.dT₁₅ micelles. Eventually, the Paclitaxel payload of ²LON-A₁₅ and ²LON-T₁₅ aggregates reached the same level (within experimental errors) upon binding the complementary oligonucleotides (compare II.2 and II.5). In other words, the dA₁₅ segment of ²LON-A₁₅ was no longer capable of promoting Paclitaxel intake once it was engaged into double-strand (ds) DNA. The importance of the dA₁₅ segment is further illustrated by the Paclitaxel loading capability of a 1:1 ²LON-A₁₅:²LON-T₁₅ mixture (FIG. 6, II.3). If the global payload of the drug increases in that case, this result is merely the result of the doubling of the effective concentration of alkyl chains. When normalized by the number of methylene groups, one clearly sees that this mixture is indeed less likely to host Paclitaxel molecules compared to ²LON-A₁₅ alone (compare white bars in II.1 and II.3). Interestingly, the drug loading capability of that mixture is within experimental errors equivalent to ²LON-T₁₅ (compare II.3 and II.4). The benefit of having the dA₁₅ sequence in ²LON-A₁₅ is therefore abolished upon annealing of the complementary oligonucleotide.

4. Controlled Release of Paclitaxel.

If ds-LONs are clearly less efficient at solubilizing Paclitaxel in aqueous buffered solutions compared to ²LON-A₁₅ or ²LON-T₁₅ micellar aggregates, the controlled release of the Paclitaxel drug molecules upon annealing of the cDNA has not been evidenced so far (FIG. 3). Despite its lower ability to host Paclitaxel molecules, these experiments were conducted with ²LON-T₁₅ micellar aggregates for their lesser tendency toward precipitation with time (see the physicochemical properties part). Schematically, the filtered micellar suspensions of ²LON-T₁₅ loaded with Paclitaxel was subjected either to the cDNA dA₁₅ or the non complementary ss-DNA (5′-GGC TCA CAA CAG GC-3′ (SEQ ID NO: 34)) as a control and the released Paclitaxel was again filtered off through 0.4 μm pores (FIG. 5). While some Paclitaxel molecules were non specifically released from the ²LON-T₁₅ micelles upon treatment with the ncDNA (ca. 2-fold decrease in Paclitaxel loading), the complementary dA₁₅ induced a 5.6-fold decrease thus demonstrating the selective controlled release of the drug.

5. Discussions.

What drives the release of the drug loaded in the hydrophobic core of the aggregates upon DNA annealing is not obvious at first sight. It must first be noticed that the nature of the LON aggregates is not changed upon binding the cDNA (FIG. 5, top) and micellar aggregates are still observed in the presence of the cDNA. Yet, contraction of the core micelle was observed following hybridization of the complementary dA₁₅ with ²LON-T₁₅ aggregates (from 14.7 to 11.8 nm). Such contraction of the micellar core was not observed with the ncDNA. These results were quite unexpected. Formation of the rather long and stirr rod-shaped dsDNA was indeed expected to increase the size of the micelle compared to the quite flexible ss-DNA segment of LONs. Consequently, the present DLS results strongly suggest that the micellar hydrophobic core experience much contraction upon DNA duplex formation. This observation may shed light on the mechanism of the triggered release of Paclitaxel from LON micelles. In fact, the ss-DNA polar heads of LONs may experience conformational freedom in the aqueous phase within the LON aggregates. The surface area occupied by each polar head of ss-LONs may be quite large. Consequently, the LON alkyl chains may be poorly packed within the hydrophobic core thus leaving room for hosting hydrophobic guests. In the contrary, the stiffness and the rather small cross section of DNA double helices might allow for a better compaction of the alkyl chains within the core of the micelle with the concomitant release of part of the loaded Paclitaxel molecules.

Conclusions

In this study the inventors present the synthesis of LONs featuring double lipophilic chains. These LONs self-assemble into micelles, which are prone to host Paclitaxel molecules within their hydrophobic cores. These results demonstrate that the composition of the LONs both in terms of the lipid and the oligonucleotide sequence impact their ability to host lipophilic molecules. Interestingly, the drug intake of ²LON-A₁₅ was markedly high compared to other LONs. Overall, the polynucleotide sequence serves three roles: (1) it promotes the transfer of the alkyl chains of the LONs into water, (2) it potentializes the drug intake of the LON aggregates and (3) it can hybridize with the cDNA sequence to trigger the release of the drug.

Numerous stimuli-bioresponsive drug delivery systems have been developed to induce the release of the drug in response to certain stimuli like pH, temperature, redox potential, enzymes, light, and ultrasound. In this contribution the inventors demonstrate that the drug loading of LON based nanomaterials can be modulated via complementary oligonucleotide stimulus. LON aggregates afford an original and specific stimuli-bioresponsive alternative to address the drug controlled delivery issue.

Example 3

This example describes the synthesis of ketal based LASOs with Phosphorothiate and LNA modifications used in Example 4.

ON1 (LASO#15 PTO):  (C15)2U*5′AAC TTG TTT CCT GCA GGT GA3′ ON2 (LASO#15 PTO Fluo):  (C15)2U*5′AAC TTG TTT CCT GCA GGT GA3′-Fluo ON3 (L#Scr PTO):  (C15)2U*5′CGT GTA GGT ACG GCA GAT C3′ ON4 (LASO#15 PTO LNA):  (C15)2U*5′AAC TTG TTT CCT GCA GGT GA3′ ON5 (LASO#15 PTO LNA Fluo):  (C15)2U*5′AAC TTG TTT CCT GCA GGT GA3′-Fluo ON6 (L#Scr PTO LNA):  (C15)2U*5′CGT GTA GGT ACG GCA GAT C3′ Oligonucleotide Synthesis.

ON1, ON2, ON3, ON4, ON5 and ON6 were synthesized using phosphoramidite methodology on an automated PerSeptive Expedite 8909 DNA synthesizer at the μmol scale on 1000 A primer support (loading: 60-100 μmol/g, Link technologies, Synbase Control Pore Glass). Prior to use, the phosphoramidite 1 was co-evaporated several times with acetonitrile, and dissolved in dry CH₂Cl₂/CH₃CN 2/1 to a 0.1 M concentration. N-benzylthiotetrazole was used for activation of phosphoramidite prior to coupling. The phosphoramidite 1 was manually coupled last on the solid support by passing (via syringes) the activator and the phosphoramidite (0.25 mL) back and forth several times for 6 min. Deblocking and detachment from the solid support was achieved using 1 mL of ammonium hydroxide solution, 28-30% for 4 h at 55° C. The supernatant was collected and the CPG beads were washed 3 times with 0.25 mL of autoclaved milliQ water. The solutions were pooled and evaporated (speed vac). The crude oligonucleotide amphiphiles ON1-ON6 were dissolved in 0.4 mL of water and purified on an semi-preparative C4-reverse phase HPLC column (Nucleosil® (Macherey & Nagel) 120-5 C4) using a linear gradient from 100% of solution A (0.1 M triethylammonium acetate, pH 7.0, 5% acetonitrile) to 100% of solution B (0.1 M triethylammonium acetate, pH 7.0, 80% acetonitrile) over 35 min, (flow rate: 5 ml/min). The product oligoamphiphiles eluted after ca. 27 min. Product containing fractions were pooled and evaporated to dryness and dissolved in autoclaved milliQ water.

MALDI-TOF mass-analyses: ON1: [M+H]⁺ calculated MW: 7183.26 Da, found: 7183.53 Da; ON2: [M+H]⁺ calculated MW: 7768.78 Da, found: 7769.65 Da; ON3: [M+H]⁺ calculated MW: 6913.02 Da, found: 6915.19 Da; ON4: [M+H]⁺ calculated MW: 7493.38 Da, found: 7508.23 Da; ON5: [M+H]⁺ calculated MW: 8078.90 Da, found: 8092.15 Da; ON6: [M+H]⁺ calculated MW: 7223.15 Da, found: 7238.74 Da.

Example 4

This example shows that the modified oligonucleotides according to the invention are capable of inhibiting TCTP expression more efficiently than the corresponding unmodified oligonucleotides.

Materials and Methods

Oligonucleotides

Different Modified Oligonucleotides were used:

-   -   ASO#15PD: DNA of sequence SEQ ID NO: 6 with a phosphodiester         backbone;     -   ASO#15PTO: DNA of sequence SEQ ID NO: 6 with a phosphorothiorate         backbone;     -   LASO#15PD: DNA of sequence SEQ ID NO: 6 with a phosphodiester         backbone bearing two hydrophobic chains at its 5′ end;     -   LASO#15PTO: DNA of sequence SEQ ID NO: 6 with a         phosphorothiorate backbone bearing two hydrophobic chains at its         5′ end;     -   ASO#15LNA: LNA of sequence SEQ ID NO: 6;     -   LASO#15LNA: LNA of sequence SEQ ID NO: 6 bearing two hydrophobic         chains at its 5′ end;     -   Scr#PD: DNA of sequence SEQ ID NO: 4 with a a phosphodiester         backbone;     -   Scr#PTO: DNA of sequence SEQ ID NO: 4 with a phosphorothiorate         backbone;     -   LScr#PD: DNA of sequence SEQ ID NO: 4 with a a phosphodiester         backbone bearing two hydrophobic chains at its 5′ end;     -   LScr#PTO: DNA of sequence SEQ ID NO: 4 with a phosphorothiorate         backbone bearing two hydrophobic chains at its 5′ end;     -   Scr#LNA: LNA of sequence SEQ ID NO: 4; and     -   LScr#LNA: LNA of sequence SEQ ID NO: 4 bearing two hydrophobic         chains at its 5′ end.         Cell Lines and Cell Culture Conditions

The CR prostate cancer cell line PC-3 was purchased from the American Type Culture Collection (Rockville, Md., USA). The cells were maintained in Dulbecco's Modified Eagle's Medium (Invitrogen, Cergy Pontoise, France), supplemented with 10% fetal calf serum (FCS).

Transfections

Lipid-conjugated oligonucleotides, LASO#15PD, LASO#15PTO and LASO#15LNA were tested on TCTP protein expression using western-blot (LScr#PD, LScr#PTO and LScr#LNA were used as negative control).

Prostatic cell line were transfected with ASO#15PD, ASO#15PTO, LASO#15PD, LASO#15PTO and LASO#15LNA at various concentrations (from 30 nM to 100 nM), in OptiMEM® medium (Invitrogen), without serum and in the presence or not of Oligofectamine™ transfection reagent (Invitrogen), according to the manufacturer's instructions.

Serum was added 4 hours after transfection. Two or three days after transfection, the level of TCTP protein expression and GAPDH (used as housekeeping gene) were determined by performing western blotting.

Western Blot Analysis

Western Blot analysis was performed as described previously (Rocchi et al., 2004, Cancer Res. 64:6595-602) with 1:2000 rabbit TCTP polyclonal antibody (Abcam Inc., Cambridge, UK). Loading levels were normalized using 1:5000 rabbit anti-glyceraldehyde-3-phosphate dehydrogenase polyclonal (Abcam Inc.).

Results

The design, synthesis and screening of ASOs targeting TCTP mRNA were performed to determine the lead sequence. Among the 27 sequences, 12 were withdrawn (lack of specificity, high GC percentage) and 15 have been tested among which, 3 have been selected (#10 of sequence SEQ ID NO: 2, #14 of sequence SEQ ID NO: 7, #15 of sequence SEQ ID NO: 6) for their efficiency to inhibit TCTP protein and cell growth.

The inventors started the chemical modification of the ASOs with the lead sequence (#15) by adding 2-3 lipid chains on the 5′ part of the sequence.

As shown in FIG. 7, they demonstrated that the addition of 2 lipid chains on the 5′ part increase the inhibition of TCTP protein, and in particular that the sequence #15 with the 2 lipid chains phosphorothioate DNA (LASO#15PTO) and phosphorothioate DNA/LNA (LASO#15LNA) are the most efficient to inhibit TCTP protein expression in the presence of Oligofectamine™.

Only the sequence #15 with the 2 lipid chains phosphorothioate was able to inhibit TCTP protein expression in absence of this transfection agent (see FIG. 8).

The inventors also demonstrated that LASO#15PTO and LASO#15LNA could penetrate the cell membrane more quickly than ASO#15PTO.

The inventors further demonstrated that the sequence #15 with the 2 lipid chains phosphorothioate DNA further had the capacity of inhibiting TCTP protein expression over a long period, since a strong to complete inhibition could be observed as late as 6 days after transfection, which was not the case with ASO#15PTO (see FIG. 9).

Example 5

FASO#15 PTO:  Flurocarbon 5′AAC TTG TTT CCT GCA GGT GA3′ This comparative example shows that the TCTP inhibition observed with the modified oligonucleotides according to the invention is not obtained with the same oligonucleotides bearing another modification. Materials and Methods Oligonucleotides Different Modified Oligonucleotides were used:

-   -   FASO#15PTO: DNA of sequence SEQ ID NO: 6 with a         phosphorothiorate backbone bearing an hydrophobic mono-chain         comprising a C₈F₁₇-moiety at its 5′ end     -   FASO#Scr: DNA of sequence SEQ ID NO: 4 with a phosphorothiorate         backbone bearing an hydrophobic mono-chain comprising a         C₈F₁₇-moiety at its 5′ end.         The modified oligonucleotides FASO#15PTO and FASO#15Scr         (structural formula below) were obtained by the “post synthetic         approach” illustrated in the schema below, following the         protocol set out in G. Godeau, H. Arnion, C. Brun, C. Staedel         and P. Barthelemy, Med. Chem. Comm., 2010, 1, 76-78, using         oligonucleotides having the sequence indicated above.

Cell Lines and Cell Culture Conditions

The CR prostate cancer cell line PC-3 was purchased from the American Type Culture Collection (Rockville, Md., USA). The cells were maintained in Dulbecco's Modified Eagle's Medium (Invitrogen, Cergy Pontoise, France), supplemented with 10% fetal calf serum (FCS).

Transfections

Lipid-conjugated oligonucleotide, FASO#15PTO was tested on TCTP protein expression using western-blot (FASO#Scr was used as negative control).

Prostatic cell line were transfected with FASO#15PTO at 100 nM, in OptiMEM® medium (Invitrogen), without serum and in the presence or not of Oligofectamine™ transfection reagent (Invitrogen), according to the manufacturer's instructions.

Serum was added 4 hours after transfection. Two or three days after transfection, the level of TCTP protein expression and GAPDH (used as housekeeping gene) were determined by performing western blotting.

Western Blot Analysis

Western Blot analysis was performed as described previously (Rocchi et al., 2004, Cancer Res. 64:6595-602) with 1:2000 rabbit TCTP polyclonal antibody (Abcam Inc., Cambridge, UK). Loading levels were normalized using 1:5000 rabbit anti-glyceraldehyde-3-phosphate dehydrogenase polyclonal (Abcam Inc.).

Results

As shown in FIG. 10, the inventors demonstrated that the oligonucleotide of sequence SEQ ID NO: 6 bearing a hydrophobic mono-chain on its 5′ part is incapable of inhibiting TCTP protein expression, in the presence or absence of Oligofectamine™.

This example thus demonstrates that the properties of the modified oligonucleotide according to the invention are not observed with oligonucleotides bearing any modification. 

The invention claimed is:
 1. A medicament comprising as an active agent, an oligonucleotide modified by substitution at the 3′ or the 5′ end by a moiety comprising at least one ketal functional group, wherein a ketal carbon of the at least one ketal functional group bears two saturated or unsaturated, linear or branched, hydrocarbon chains comprising from 1 to 22 carbon atoms, wherein said oligonucleotide is an antisense oligonucleotide or an interfering RNA which targets an mRNA of interest and is capable of reducing the amount of protein encoded by said mRNA.
 2. The medicament according to claim 1, wherein the modified oligonucleotide is of the general formula (I):

wherein: Oligo represents an oligonucleotide sequence which may be oriented 3′-5′ or 5′-3′, simple and/or double stranded, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and/or comprise modified nucleotides selected from the group consisting of locked nucleic acid (LNA), 2′-OMe analogs, 2′-phosphorothioate analogs, 2′-fluoro analogs, 2′-Cl analogs, 2′-Br analogs, 2′-CN analogs, 2′-CF₃ analogs, 2′-OCF₃ analogs, 2′-OCN analogs, 2′-O-alkyl analogs, 2′-S-alkyl analogs, 2′-N-alkyl analogs, 2′-O-alkenyl analogs, 2′-S-alkenyl analogs, 2′-N-alkenyl analogs, 2′-SOCH₃ analogs, 2′-SO₂CH₃ analogs, 2′-ONO₂ analogs, 2′-NO, analogs, 2′-N₃ analogs, 2′-NH, analogs and combinations thereof; X represents a divalent linker moiety selected from ether —O—, thio —S—, amino —NH—, and methylene —CH₂—; R₁ and R₂ may be identical or different and represent: (i) a hydrogen atom, (ii) a halogen atom, (iii) a hydroxyl group, (iv) an alkyl group comprising from 1 to 12 carbon atoms; L₁ and L₂ may be identical or different and represent a saturated or unsaturated, linear or branched hydrocarbon chain comprising from 1 to 22 carbon atoms, B is an optionally substituted nucleobase, selected from the group consisting of purine nucleobases, pyrimidine nucleobases, and non-natural monocyclic or bicyclic heterocyclic nucleobases wherein each cycle comprises from 4 to 7 atoms.
 3. The medicament according to claim 2, wherein the modified nucleotides are selected from the group consisting of LNA, 2′-OMe analogs, 2′-phosphorothioate analogs and 2′-fluoro analogs.
 4. The medicament according to claim 1 wherein the oligonucleotide is selected from the group consisting of dT₁₅, dA₁₅, and an oligonucleotide consisting of the sequence SEQ ID NO:
 6. 5. The medicament according to claim 2, wherein the divalent linker moiety is ether —O—.
 6. The medicament according to claim 2, wherein R₁ and R₂ are hydrogen atoms.
 7. The medicament according to claim 2, wherein L₁ and L₂ represent a hydrocarbon chain comprising from 6 to 22 carbon atoms.
 8. The medicament according to claim 2, wherein B is a non substituted nucleobase selected from the group consisting of uracil, thymine, adenine, cytosine, 6-methoxypurine and hypoxanthine.
 9. The medicament according to claim 2, wherein the oligonucleotide comprises a fragment of at least 10 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO: 2 (5′-ACCAATGAGCGAGTCATCAA-3′), SEQ ID NO: 3 (5′-AACCCGUCCGCGAUCUCCCGG-3′), SEQ ID NO: 6 (5′-AACTTGTTTCCTGCAGGTGA-3′),  SEQ ID NO: 7 (5′-TGGTTCATGACAATATCGAC-3′), SEQ ID NO: 8 (5′-TAATCATGATGGCGACTGAA-3′), SEQ ID NO: 16 (5′-ACCAGTGATTACTGTGCTTT-3′),  SEQ ID NO: 17 (5′-CTTGTAGGCTTCTTTTGTGA-3′), SEQ ID NO: 18 (5′-ATGTAATCTTTGATGTACTT-3′), SEQ ID NO: 19 (5′-GTTTCCCTTTGATTGATTTC-3′),  SEQ ID NO: 20 (5′-TTCTGGTCTCTGTTCTTCAA-3′), SEQ ID NO: 25 (5′-AGAAAATCATATATGGGGTC-3′), SEQ ID NO: 27 (5′-TTAACATTTCTCCATTTCTA-3′),  SEQ ID NO: 29 (5′-GTCATAAAAGGTTTTACTCT-3′) and  SEQ ID NO: 31 (5′-GAAATTAGCAAGGATGTGCT-3′).


10. The medicament according to claim 1, wherein the oligonucleotide comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO:
 7. 11. A method for treating cancer, comprising the administration to a patient in need thereof of a pharmaceutically acceptable amount of a medicament according to claim
 1. 12. The method of claim 11 wherein the cancer is selected from the group consisting of prostate cancer, colon cancer, colorectal cancer, breast cancer, liver cancer, erythroleukemia, gliomas, melanomas, hepatoblastomas and lymphomas.
 13. The method of claim 11 wherein the cancer is a prostate cancer.
 14. An aqueous composition comprising oligonucleotides modified by substitution at the 3′ or the 5′ end by a moiety comprising at least one ketal functional group, wherein a ketal carbon of the at least one ketal functional group bears two saturated or unsaturated, linear or branched, hydrocarbon chains comprising from 1 to 22 carbon atoms, wherein the modified oligonucleotides self-assembled into micelles, and optionally comprising a hydrophobic active principle hosted in said micelles.
 15. The aqueous composition according to claim 14 as a vehicle.
 16. The aqueous composition of claim 15, as a vehicle of a sparingly hydrosoluble active principle.
 17. A medicament comprising the aqueous composition as defined in claim
 14. 18. A method for treating cancer, comprising the administration to a patient in need thereof of a pharmaceutically acceptable amount of the aqueous compositions according to claim
 14. 19. The aqueous composition according to claim 14, wherein said hydrophobic active principle is selected from the group consisting of Paclitaxel, Docetaxel, Vincristine, Vinorelbine, and Abraxane; Tamoxifen, Gonadotrophin-releasing hormone (GnRH) agonists and antagonists, androgen receptor (AR) antagonist, and estrogen receptor (ER) antagonists; Cyclophosphamide, Chlorambucil and Melphalan; Methotrexate, Cytarabine, Fludarabine, 6-Mercaptopurine and 5-Fluorouracil; Doxorubicin, Irinotecan, Platinum derivatives, Cisplatin, Carboplatin, Oxaliplatin; Bicalutamide, Anastrozole, Examestane and Letrozole; Imatinib (Gleevec), Gefitinib and Erlotinib; Rituximab, Trastuzumab (Herceptin) and Gemtuzumab ozogamicin; Interferon-alpha; Tretinoin and Arsenic trioxide; Bevicizumab, Serafinib and Sunitinib.
 20. The aqueous composition according to claim 16, wherein said sparingly hydrosoluble active principle is selected from the group consisting of Paclitaxel, Docetaxel, Vincristine, Vinorelbine, and Abraxane; Tamoxifen, Gonadotrophin-releasing hormone (GnRH) agonists and antagonists, androgen receptor (AR) antagonist, and estrogen receptor (ER) antagonists; Cyclophosphamide, Chlorambucil and Melphalan; Methotrexate, Cytarabine, Fludarabine, 6-Mercaptopurine and 5-Fluorouracil; Doxorubicin, Irinotecan, Platinum derivatives, Cisplatin, Carboplatin, Oxaliplatin; Bicalutamide, Anastrozole, Examestane and Letrozole; Imatinib (Gleevec), Gefitinib and Erlotinib; Rituximab, Trastuzumab (Herceptin) and Gemtuzumab ozogamicin; Interferon-alpha; Tretinoin and Arsenic trioxide; Bevicizumab, Serafinib and Sunitinib. 